The plasma-membrane monoamine transporters (MATs), including the serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, serve a pivotal role in limiting monoamine-mediated neurotransmission through the reuptake of their respective neurotransmitters. The transporters are the main target of clinically used psychostimulants and antidepressants. The continuing need for therapeutic drugs to treat brain disorders involving aberrant monoamine signaling provides a compelling reason to further our understanding of transporter function and to identify novel ways of targeting them. We propose to characterize a novel modulatory site on SERT, NET, and DAT, using computational modeling and functional studies. Furthermore, we will employ this site in virtual screens to identify transporter-interacting compounds with novel transporter-modulating activities. Our approach is supported by our earlier studies on monoamine transporters from the human parasite Schistosoma mansoni in which we identified structures outside the translocation pathway in SERT to be important for ligand interaction. We have successfully employed the allosteric site in a virtual drug screen and we have identified several interacting compounds with unique transporter-modulating activities. The activities we have identified are 1) uptake enhancement; and 2) allosteric interference with transporter/cocaine interaction. In this proposal we will in aim 1 refine and characterize this modulatory pocket in all three transporters employing computational modeling. We will also employ structure/function studies to provide insight into how the modulatory compounds interact with the allosteric site and produce their modulatory activities. We will use this knowledge to inform additional virtual drug screens to identify MAT modulators that display high efficacy, potency, and selectivity. In aim 2 we will design and synthesize compounds based on our initial hits to establish structure/activity relationships of these compounds. We will employ these unique compounds and establish their mechanism of action through structure/function studies. We will focus on how conformational transitions are modulated and how the modulatory site affects the orthosteric substrate binding site. Behavior modulating activity of the compounds will be established in planarian-based assays. Successful completion of this project will lead to important insights into novel ways of regulating monoamine transporter action. We specifically expect to identify molecules that enhance transporter function and alter the interaction between the transporters and their classical ligands, including psychostimulants and antidepressants. The work will also supply probes that can provide mechanistic information about monoamine transporter function. Importantly this work will position us to better understand how this novel allosteric site within te MATs can be employed as a therapeutic target that could lead to more effective treatment of mental disorders that involve monoamine signaling, including depression, ADHD, schizophrenia, impulse control disorders, and drug and alcohol abuse.